The Role of a Complex Microbial Community in Supporting Dehaloccoides Bioaugmentaion
Samuel Fogel, Bioremediation Consulting, Inc., Watertown, MA

In Situ Metals Remediation with Metal Remediation Compound (MRC®)
Anna Willett, Regenesis, San Clemente, CA

Degradation of RDX with Reduced Humic Substances
Man Jae Kwon, University of Illinois - Urbana Champaign, Urbana, IL

Enhanced Microbial Consortium Production in a Slurry Bioreactor for Creosote-Contaminated Soil Bioaugmentation Using Humic Acids
Dominic D'Amours, Ecole Polytechnique de Montreal, Montreal, QC, Canada

Identification of Tetrachloroethene- and Vinyl Chloride-Dechlorinating Bacteria Enriched From Tetrachloroethane-Contaminated Groundwater and Sediments
Eun-Kyeu Son, Rutgers University, New Brunswick, NJ

EMCÔ for In Situ Bioremediation of Groundwater Containing Chloroethanes and other Chlorinated Solvents
Jim Mueller, Adventus Americas Inc., Bloomingsale, IL  

Mutagenicity of a Former Gasworks Soil During Bioremediation

Paul A. White, Mutagenesis Section, Safe Environments Programme, Health Canada, Tunney’s Pasture 0803A, Ottawa, ON K1A 0L2, Tel: 613-941-7373, Fax: 613-941-8530, Email: paul_white@hc-sc.gc.ca
Krista D. Lynes, Mutagenesis Section, Safe Environments Programme, Health Canada, Tunney’s Pasture 0803A, Ottawa, ON K1A 0L2, Tel: 613-957-3135, Fax: 613-941-8530, Email: krista_lynes@hc-sc.gc.ca
Staffan Lundstedt, Department of Chemistry, University of Umeå, SE-90187, Umeå, Sweden, Tel: 90-7866654, Fax: 90-128133, Email: staffan.lundstedt@chem.umu.se
Lars Öberg, Department of Chemistry, University of Umeå, SE-90187, Umeå, Sweden, Tel: 90-7867622, Fax: 90-128133, Email: lars.oberg@envichem.umu.se
George R. Douglas, Mutagenesis Section, Safe Environments Programme, Health Canada, Tunney’s Pasture 0803A, Ottawa, ON K1A 0L2, Tel: 613-957-3137, Fax: 613-941-8530, Email: george_douglas@hc-sc.gc.ca 
Iain B. Lambert, Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, Canada, K1S 5B6, Tel: 613-520-2600 x3893, Fax: 613-520-3539, Email: iainlambert@pigeon.carleton.ca

Although bioremediation is often assumed to be a detoxifying process, the production of degradation products can increase potential hazard.  This study followed a pilot scale (750 L) bioslurry remediation of a PAC-contaminated soil from an aged gasworks site near Stockholm, Sweden. Chemical and biological analyses followed changes in the concentrations of PACs and PAC derivatives, and mutagenic activity at remediation days 0, 3, 7, 24 and 29.  Sample extraction and fractionation employed pressurized fluid extraction and subsequent elution from partially deactivated silica. Two fractions (2 & 3), enriched in non-polar neutral (e.g., alkyl-PACs) and polar aromatic (e.g., N-heterocyclic) compounds, were analysed for mutagenic activity using the plate incorporation version of the Salmonella mutagenicity test on strains TA98, TA100, YG1041 and YG1042.  The metabolically enhanced YG strains permitted enhanced detection of mutagenic nitroarenes and aromatic amines.  Thorough chemical analysis revealed noteworthy carcinogenic PACs including benz[a]anthracene, benzo[a]pyrene and dibenz[ah]anthracene, as well as other PACs, alkyl-PACs, and O- and S-heterocyclics in fraction 2. Fraction 3 contained a variety of N-heterocyclic compounds and oxy-PACs.  The heavier PACs, such benzo[b]fluoranthene, showed modest declines (i.e., 14%) over the course of the remediation, while lighter PACs, such as anthracene, showed large reductions in concentration (i.e., 86%).  Oxy-PACs, such as 4-oxapyrene-5-one and 1-acenaphthenone, increased in concentration during the remediation (i.e., 29% and 56%, respectively).  The mutagenicity results showed a net 2- to 8-fold increase in frameshift mutagenic activity for the non-polar neutral fraction, and a 7- to 32-fold increase in the activity of the polar aromatic fraction.  The patterns of mutagenic activity strongly suggest the production and accumulation of hitherto unidentified N-heterocyclics and/or other aromatic amines.  Mutagenicity assessment of the identified compounds (currently underway) will determine the degree to which known compounds can account for the observed biological activity.

In Situ Metals Remedation with Metals Remedation Compound (MRC^®)

Anna Willett, Regenesis, 1011 Calle Sombra, San Clemente, CA  92673; Tel: 949-366-8000, Fax: 949-366-8090, Email: awillett@regenesis.com
Stephen S. Koenigsberg, Regenesis, 1011 Calle Sombra, San Clemente, CA  92673; Tel: 949-366-8000, Fax: 949-366-8090, Email: skoenigsberg@regenesis.com

Contamination of groundwater by metals has not been widely addressed by engineered in situ remediation technologies, despite the documentation of metals contamination at greater than 50% of sites from the National Priorities List and at Department of Defense and Department of Energy locations.  Metals Remediation Compound (MRC® is a slow-release metals remediation product that removes dissolved metals from groundwater via in situ immobilization (precipitation and/or sorption to soil particles).  The immobilized metals are stable under reducing conditions and may be stable under oxidizing conditions, depending on the identity of the metal and site specific geochemistry. 

MRC consists of an organosulfur compound esterified to a carbon backbone.  This organosulfur ester is embedded in a polylactate matrix, making MRC a thick, viscous liquid.  Upon injection into an aquifer, the organosulfur compound is slowly released when the ester bonds in MRC are cleaved via hydrolysis by water and microbial enzymatic action.  The organosulfur moiety interacts with metal ions, either to complex them or to reduce them and complex them sequentially.  These complexes sorb strongly to soil, filter media, or other solid supports.  MRC also slowly releases lactate, which acts as an electron donor and carbon source for naturally occurring bacteria and creates the optimal conditions for metals immobilization by the organosulfur compound.  For sites with mixed metal and chlorinated solvent contamination, MRC provides a substrate for accelerated reductive dechlorination and metals immobilization.

MRC’s ability to remove dissolved metals, such as arsenic, copper, chromium, cadmium, mercury, and lead, from solution has been tested in the laboratory and verified in situ via injection into metals-contaminated aquifers.  Additionally, detailed modeling and kinetics calculations have been performed to investigate the stability of precipitated metals.  Results from theoretical studies, as well as field applications for a variety of metals will presented. 

Degradation of RDX with Reduced Humic Substances

Man Jae Kwon, University of Illinois - Urbana Champaign, Dept of Civil and Environmental Engineering, NCEL 205 N. Mathews, Urbana, IL, 61801, Tel: 217-333-6851, Fax: 217-333-6967, Email: mankwon@uiuc.edu
Kevin T. Finneran, University of Illinois - Urbana Champaign, Dept of Civil and Environmental Engineering, NCEL 205 N. Mathews, Urbana, IL, 61801, Tel: 217-333-1514, Fax: 217-333-6967, Email: finneran@uiuc.edu

Previous studies have indicated that humic substances and Fe(II) can abiotically transfer electrons to a variety of compounds. These include nitroaromatic and nitramine compounds, which can be reduced to less harmful metabolites. In this study, the potential for reduced humic substances and Fe(II) to degrade hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was evaluated.

50 mM RDX was anaerobically incubated with 1mM, 500 mM, and 100 mM of chemically reduced AQDS (2,6-anthraquinone disulphonate) and biologically reduced AQDS, an analog for humic substances. RDX reduction was also investigated with 1.2 mM and 600 mM of soluble Fe(II). 

RDX was completely degraded within 5 hours and 7 hours with 1mM and 500 mM of both of reduced AQDS, respectively. 100 mM of reduced AQDS reduced RDX to approximately 20 mM in 12 hours. Reduced AQDS transfers two electrons per mole in coupled oxidation/reduction reaction. RDX accepts six electrons per mole. Therefore three times as much reduced AQDS is needed to completely reduce RDX. Incubating the RDX with 100 mM of reduced AQDS resulted in reduction of two thirds of the 50 mM RDX. This was the predicted stoichiometry and demonstrates that RDX degradation is due to humics-mediated electron transfer. 50 mM RDX, however, was only reduced to 19 mM and 39 mM with 1.2 mM and 600 mM of soluble Fe(II) for 52 hours, respectively. The results of this study demonstrate that reduced AQDS can transfer electrons directly to RDX and will degrade RDX faster than soluble Fe(II).

These findings suggest that reduced humic substances react directly with RDX. This may eventually be used as a rapid and effective cleanup strategy in both Fe(III) rich and Fe(III) poor environments. Upcoming experiments include sediment incubations and cell suspensions of Fe(III)- and humics-reducing Bacteria (e.g. Geobacter metallireducens) to better evaluate anaerobic biodegradation of RDX under in situ conditions.

Enhanced Microbial Consortium Production in a Slurry Bioreactor for Creosote-Contaminated Soil Bioaugmentation Using Humic Acids

Dominic D’Amours, NSERC Industrial Chair in Site Remediation and Management, École Polytechnique de Montréal, Chemical Engineering Department, P.O. Box 6079, Station “Centre-ville”, Montreal, Quebec, Canada H3C 3A7, Tel: 514-340-4711 ext 4794, Fax: 514-340-5913, Email: dominic.damours@polymtl.ca
Réjean Samson, NSERC Industrial Chair in Site Remediation and Management, École Polytechnique de Montréal, Chemical Engineering Department, P.O. Box 6079, Station “Centre-ville”, Montreal, Quebec, Canada H3C 3A7, Tel: 514-340-4898, Fax: 514-340-5913, Email: rejean.samson@polymtl.ca
Louise Deschênes, NSERC Industrial Chair in Site Remediation and Management, École Polytechnique de Montréal, Chemical Engineering Department, P.O. Box 6079, Station “Centre-ville”, Montreal, Quebec, Canada H3C 3A7, Tel: 514-340-5974, Fax: 514-340-5913, Email: louise.deschenes@polymtl.ca

The widespread contamination of soil by creosote in industrialized countries has created the need for reliable and cost-effective bioremediation processes.  Polycyclic aromatic hydrocarbons (PAHs) in creosote are of particular concern because they represent up to 85% of its weight composition and many are carcinogenic and degrade poorly.  Soil activation, a method based on the cultivation of a microbial consortium from a fraction of a contaminated soil for subsequence use as an inoculum for bioaugmentation of the same soil, was studied as a method for the bioremediation of creosote-contaminated soils.  Since the bioavailability of PAHs is low due to their high hydrophobicity, humic acids were added during soil activation to increase it.  The effect of this non-toxic biogenic material on the microbial consortium performance was investigated.  An indigenous microbial consortium capable of degrading the PAH fraction of creosote was produced in 8 L stainless steel soil slurry (10% w/v) bioreactors.  The bioreactors were operated in fed-batch mode with periodic creosote addition in increasing quantity (0.2 to 3.2 mL/L).  Eight of the twelve bioreactors were supplemented with humic acids to test their influence on soil activation.  During the 60-day period of activation, PAHs, pH, temperature and dissolved oxygen levels were monitored.  Microbial community performance was monitored using mineralization tests, direct counts using Live/Dead® BacLight^TM method, PAH-specific most probable number (MPN) bacterial counts, and denatured gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA.  The presence of humic acids increased PAH degradation and mineralization rates resulting in an enhanced microbial consortium performance.  The presence of higher concentration of PAH-degraders and an enhanced microbial tolerance to increasing concentration of creosote were also attributed to the addition of humic acids.  These results indicate that the use of humic acids during creosote-contaminated soil bioactivation efficiently enhances microbial consortium performance.

Identification of Tetrachloroethene- and Vinyl Chloride-Dechlorinating Bacteria Enriched from Tetrachloroethene-Contaminated Groundwater and Sediments

Eun-Kyeu Son, Environmental Sciences, Rutgers University, 14 College Farm Road, New Brunswick, NJ, Tel: 732-932-4961, Fax: 732- 932-8644, Email: eunkyeu@eden.rutgers.edu
Kenneth Y. Lee, Civil & Environmental Engineering, Rutgers University, 623 Bowser Road, Piscataway, NJ, Tel: 732-445-2240, Fax: 732-445-0577, Email: kenlee@rci.rutgers.edu
Donna E. Fennell, Environmental Sciences, Rutgers University, 14 College Farm Road, New Brunswick, NJ, Tel: 732-932-4961, Fax: 732- 932-8644, Email: fennell@envsci.rutgers.edu

We identified the dechlorinating bacteria enriched from a tetrachloroethene (PCE)-contaminated fractured rock aquifer at Rutgers University in Piscataway, NJ. This site is part of the Brunswick (Passaic) formation of the Newark Basin that makes up of much of northern NJ. Five sets of enrichments using groundwater containing 1% sediment fines were established (killed controls, live controls, electron donor only, electron donor + PCE, and electron donor + vinyl chloride (VC)). Chloroethenes and methane were determined and PCE, VC and electron donor (butyrate) were re-amended periodically. Enrichments were transferred over the course of two years. Complete dechlorination of chloroethenes to ethene was detected in all electron donor-amended bottles. Little dechlorination was observed in the live controls not receiving electron donors and or in the autoclaved controls. These results imply that the dechlorination is attributed to microbially mediated reduction and that the aquifer is electron donor limited. Molecular characterization was carried out using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). At least two different Dehalococcoides-like organisms were detected in second generation PCE- and VC-amended cultures by DGGE. A distinct band with a DNA sequence with high similarity to members of the Geobacteraceae was also present in PCE-amended cultures. PCR using primers specific for published dehalogenases yielded bands of the expected size for a PCE dehalogenase (PceA Y51 from Desulfitobacterium sp.Y51) and a TCE dehalogenase (TceA from Dehalococcoides ethenogenes 195) in PCE-amended cultures. PCR with primers specific for two VC dehalogenases (BvcA from Dehalococcoides sp. BAV1 and VcrA from Dehalococcoides sp. VS1) yielded bands of the expected size in both PCE and VC amended cultures. These findings suggest that the aquifer harbors a diverse chloroethene-dechlorinating community.

EHC^TM for In Situ Bioremediation of Groundwater Containing Chloroethanes and other Chlorinated Solvents

Jim Mueller, Adventus Americas Inc., 109 Fairfield Way, Suite 207, Bloomingdale, IL 60108, Tel: 630-295-8661, Fax: 630-295-8664, Email: jmueller@adventus.us 
John Vogan, EnviroMetal Technologies Inc. 745 bridge street west Suite 7 Waterloo, Ontario, Canada, Tel: 519-746-2204 ext 24, Email: jvogan@eti.ca 
David Hill, Eva Dmitrovic, and Alan Seech, Adventus Remediation Technologies Inc., 1345 Fewster Drive, Mississauga, Ontario, Canada, Tel: 905-273-5374 Ext. 221, Fax: 905-273-4367, Email: alan.seech@adventustech.com

EHC^TM bioremediation products uniquely combine various organic carbon sources with zero valent iron (ZVI) and/or other reduced metals to stimulate biological activity and direct reduction of organic compounds that are notoriously recalcitrant to biodegradation processes.  We validated the ability of EHC to remove a mixture of chlorinated volatile organic compounds (CVOCs) from groundwater; 1,1,2-Trichloroethane (1,1,2-TCA), 1,2-dichloroethane (1,2-DCA) cis-1,2-Dichloroethylene (cis-1,2-DCE), vinyl chloride (VC), chloroform (CF), trichloroethylene (TCE), carbon tetrachloride (CT), tetrachloroethylene (PCE) and dichloromethane DCM.  Following 62 days of continuous flow conditions, the concentration of 1,2-DCA decreased from 329,000 to 19 mg/L which corresponded to >99% removal of 1,2-DCA.  Removal of all other compounds was also observed, yielding a total CVOC reduction of >99%.  A chloride mass balance was conducted yielding excellent correlation between theoretical chloride concentration and CVOC removal.

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